Recently, we have investigated the mechanism of diffusion-limited binding of an intrinsically disordered protein (IDP), the N-terminal transactivation domain (TAD) of p53, to one of its binding partners, the nuclear coactivator binding domain (NCBD) of CBP. Diffusion-limited association of IDPs is a counterintuitive phenomenon because it suggests that a disordered protein should fold almost as soon as it encounters with a binding target, which seems very unlikely. We found that a transient complex (TC), which appears during binding, is unexpectedly long lasting (lifetime of several hundred microsecond) due to the stabilization by non-native electrostatic interactions. The long lifetime of TC allows for unstructured TAD to fold without dissociation once it encounters with NCBD, which makes diffusion-limited association possible. Although we have focused on the diffusion-limited association in this study, our experimental observation suggests that the formation of TC by non-native interactions generally occur in IDP binding even for slower binding systems. The next step in this project is to investigate more detailed molecular mechanism of binding. For example, binding pathways are supposed to be heterogeneous as observed in MD simulations. To probe and analyze diverse binding pathways, we will use three-color FRET. By attaching the third dye to the binding partner it will be possible to detect conformational changes of IDPs and the interaction with binding partners at the same time. So far, we have developed three-color FRET and fluorescence lifetime analyses that can probe slow processes such as oligomerization of the tetramerization domain of the tumor suppressor protein p53 (published in PNAS 2017). Currently, we are developing a fast three-color FRET method in collaboration with Dr. Irina V. Gopich at LCP. The improvement of the time resolution will allow for the investigation of microsecond millisecond dynamics conformational dynamics of various systems. This development of single molecule fluorescence method will also be useful to study oligomerization of proteins that eventually for amyloid fibrils. As a first step in this project, we investigated monomer conformations of amyloid beta protein that is associated with Alzheimers disease. The monomer conformation of this protein has been studied using various methods including nuclear magnetic resonance (NMR) and molecular dynamics (MD) simulations. The consensus of conclusions of these studies is that the monomer is disordered. However, there is a controversy over the existence of residual structures that may be templates of the fibril formation. To address this controversy, we studied the two most abundant isoforms, the 40-residue (Abeta40) and 42-residue (Abeta42) peptides. Single molecule FRET experiments have a great advantage in studying Abeta monomers because the experiment is carried out at a very low protein concentration without complications from oligomer formation or aggregation. We observed that both isoforms behave like intrinsically disordered proteins with a characteristic conformational fluctuation time of 40 ns. In collaboration with Dr. Robert B. Bests group, we have complemented the FRET experiments with MD simulations using newly-developed force fields that properly characterize the dimension and dynamics of intrinsically disordered proteins. Both the experiments and simulations clearly show that both Abeta isoforms are largely disordered and there is an almost complete absence of states with stable secondary structures.